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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Chemicals, peptides, and recombinant proteins
Triethylammonium bicarbonate
Sigma
Catalog: T7408
Phosphatase Inhibitor Cocktail 2
Sigma
Catalog: P5726
Phosphatase Inhibitor Cocktail 3
Sigma
Catalog: P0044
BCA Protein Assay Kit
Thermo Scientific Pierce
Catalog: 23225
Sodium deoxycholate
FUJIFILM Wako
Catalog: 190-08313
Sodium lauroyl sarcosinate
FUJIFILM Wako
Catalog: 198-14745
Iron-(III) chloride
FUJIFILM Wako
Catalog: 091-00872
Dithiothreitol
Thermo Scientific
Catalog: 20291
Iodoacetamide
Thermo Scientific
Catalog: A3221
Lysyl endopeptidase
FUJIFILM Wako
Catalog: 129-02541
Sequencing-grade modified trypsin
Promega
Catalog: V517
Ni-NTA silica resins
QIAGEN
Catalog: 31314
Empore SDB-XC membrane disks
CDS
Catalog: 13-110-020
Titanium dioxide (10 mm)
GL Sciences
Catalog: 5020-75010
CK2a2/b (CSNK2A2/B)
Carna Biosciences
Catalog:05-185
PKACa(PRKACA)
Carna Biosciences
Catalog:01-127
ERK2 (MAPK1)
Carna Biosciences
Catalog:04-143
EGFR (ERBB1)
Carna Biosciences
Catalog:08-115
SRC
Carna Biosciences
Catalog:08-173
JNK1(MAPK8)
Carna Biosciences
Catalog:04-163
CDK1 (CDC2/CycB1)
Carna Biosciences
Catalog:04-102
p38a(MAPK14)
Carna Biosciences
Catalog:04-152
TMTsixplexTM
Thermo Scientific
Catalog:90061
https://zenodo.org/
https://doi.org/10.5281/zenodo.5750874
ATCC
Catalog: CCL-2.2
MaxQuant
PMID: 27809316
https://www.maxquant.org/
Perseus
PMID: 27348712
https://maxquant.net/perseus/
Deposited data
Zenodo
Experimental models: Cell lines
HeLa S3
Software and algorithms
RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yasushi
Ishihama (yishiham@pharm.kyoto-u.ac.jp).
Materials availability
This study did not generate new unique reagents.
Data and code availability
Data described in this paper have been deposited at https://zenodo.org and are publicly available as of the date of publication.
DOIs are listed in the key resources table.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request
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EXPERIMENTAL MODEL AND SUBJECT DETAILS
HeLa S3 cells were cultured in DMEM containing 10% fetal bovine serum and 100 mg/mL kanamycin. For isobaric acidophilic motifcentric phosphoproteomes, cells were not stimulated (mock) or were stimulated with 10 mM CK2 inhibitor (CX-4945) for 30 min. For
isobaric basophilic motif-centric phosphoproteomes, cells were not stimulated (mock) or were stimulated with 10 mM PKA activator
(forskolin) for 30 min. For isobaric tyrosine and multiple motif-centric phosphoproteome, cells were treated with 10 mM EGF, 10 mM
EGF/10 mM afatinib, and 500 mM PV (pH 10 with 0.14% H2O2), respectively, for 30 min before harvesting. Two biological replicates
were performed.
METHOD DETAILS
Tryptic peptides from HeLa cell lysate
Cells were washed three times with ice-cold phosphate-buffered saline (phosphate-buffered saline, 0.01 M sodium phosphate,
0.14 M NaCl, pH 7.4) and then lysed in lysis buffer containing 12 mM sodium deoxycholate, 12 mM sodium lauroyl sarcosinate in
100 mM triethylammonium bicarbonate. Protein concentration was determined by means of BCA protein assay. The lysates were
digested based on the reported phase-transfer surfactants protocol (Masuda et al., 2008). The digested peptides were desalted
on SDB-XC StageTips (Rappsilber et al., 2007).
In vitro kinase reactions
For acidophilic, Pro-directed and tyrosine kinase reactions, the tryptic peptides were dissolved in 40 mM Tris-HCl (pH 7.5) and incubated with each kinase (0.2 mg CK2, ERK2, JNK1, p38a, CDK1 or SRC) at 37 C overnight for in vitro kinase reactions in the presence
of 1 mM ATP and 20 mM MgCl2. For the EGFR kinase reactions, tryptic peptides were firstly passed through SCX StageTips (Rappsilber et al., 2007) to remove afatinib. Eluted peptides were further desalted on SDB-XC StageTips. Then, the desalted peptides
were dissolved in 40 mM Tris-HCl (pH 7.5) and incubated with EGFR (0.2 mg) for in vitro kinase reactions in the presence of 1 mM
ATP and 4 mM MnCl2 at 37 C overnight. For basophilic kinases such as PKA, the lysates were loaded onto a 10-kDa ultrafiltration
device (Amicon Ultra, Millipore). The device was centrifuged at 14,000 g to remove the detergents. Subsequently, the original lysis
buffer was replaced with 40 mM Tris-HCl (pH 7.5) followed by centrifugation. Then, the proteins were incubated with 0.2 mg PKA for
in vitro kinase reactions in the presence of 1 mM ATP and 20 mM MgCl2 at 37 C overnight. After the kinase reactions, the proteins
were reduced with 10 mM DTT for 30 min at 37 C and alkylated with 50 mM iodoacetamide in the dark for 30 min at 37 C. The resulting samples were digested by Lys-C (1:100, w/w) at 37 C for 3 h followed by trypsin (1:50, w/w) overnight at 37 C. All the peptides
were desalted on SDB-XC StageTips.
TMT labeling for digested peptides
The desalted peptides were dissolved in 200 mM HEPES (pH 8.5). Then, the resuspended digested peptides were mixed with TMT
reagent dissolved in 100% ACN for 1 h. The labeling reaction was stopped by adding 5% hydroxylamine for 15 min, followed by acidification with TFA. All the peptides labeled with each multiplexed TMT reagent were mixed into the same tube and the mixture was
diluted to decrease the concentration of ACN to less than 5%. The TMT-labeled peptides were desalted on SDB-XC StageTips. The
information on the peptide amount in each TMT channel for all experiments is shown in Table S3. Note that the peptide amount for
each TMT channel was quantified by means of nanoLC-UV at 210 nm using a Thermo Ultimate 3000 RSLCnano system (Germering),
an MU701 UV detector (GL Sciences), and a C18 analytical column (150 mm length 3 100 mm ID) packed with Reprosil-Pur 120 C18AQ material (3 mm, Dr. Maisch).
IMAC
The procedure for phosphopeptides purification with an Fe3+-IMAC tip was as described previously (Tsai et al., 2014, 2015) with minor modifications. In brief, a buffer consisting of 50 mM EDTA in 1 M NaCl was used for removing Ni2+ ions. Then, the metal-free NTA
was activated by loading 100 mM FeCl3 into the IMAC tip. The Fe3+-IMAC tip was equilibrated with 0.5% (v/v) acetic acid at pH 3.0
before sample loading. Tryptic peptides from HeLa lysates were reconstituted in 0.5% (v/v) acetic acid and loaded onto the IMAC tip.
After successive washing steps with 1% (v/v) TFA in 80% ACN and 0.5% (v/v) acetic acid, the IMAC tip was coupled to an activated
SDB-XC StageTip and the bound phosphopeptides were eluted onto the SDB-XC StageTip with 200 mM NH4H2PO4 buffer. Then, the
eluted phosphopeptides were desalted with SDB-XC StageTip.
LC-MS/MS analysis
NanoLC-MS/MS analyses were performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific), which was
connected to the Thermo Ultimate 3000 RSLCnano system and an HTC-PAL autosampler (CTC Analytics). Peptide mixtures were
loaded onto and separated on self-pulled needle columns (150 mm length 3 100 mm inner diameter) packed with Reprosil-Pur
120 C18-AQ material (3 mm) or a 2-m-long C18 monolithic silica capillary column (Iwasaki et al., 2010). The mobile phases consisted
of (A) 0.5% acetic acid and (B) 0.5% acetic acid and 80% acetonitrile. Peptides were separated through a gradient from 17.5 % to
45% buffer B at a flow rate of 500 nL/min. Full-scan spectra were acquired at a target value of 43105 with a resolution of 60,000. Data
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were acquired in a data-dependent acquisition mode using the top-speed method (3 s). The peptides were isolated using a quadrupole system (the isolation window was 0.7). The MS2 analysis was performed in the ion trap using collision-induced dissociation
fragmentation with a collision energy of 35 at a target value of 1 3 104 with 100 ms maximum injection time. The MS3 analysis was
performed for each MS2 scan acquired by using multiple MS2 fragment ions isolated by an ion trap as precursors for the MS3 analysis
with a multinotch isolation waveform (McAlister et al., 2014). HCD fragmentation was used for MS3 scan with an NCE of 65%, and the
fragment ions were detected by the Orbitrap (resolution 15,000). The AGC target was 53104 with a maximum ion injection time of
22 ms. The raw data sets have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.
org) via the jPOST partner repository (https://jpostdb.org) (Moriya et al., 2019) with the dataset identifier JPST001027 (PXD026996).
Data analyses
Database search. The raw MS/MS data were processed with MaxQuant (Cox and Mann, 2008; Tyanova et al., 2016a). Peptide
search with full tryptic digestion and a maximum of two missed cleavages was performed against the SwissProt human database
(20,102 entries). The mass tolerance for precursor and MS3 ions was 4.5 ppm, whereas the tolerance for MS2 ions was 0.5 Th.
Acetylation (protein N-terminal), oxidation (M) and phospho (STY) were set as variable modifications and carbamidomethyl (C) was
set as a fixed modification. The quantitation function of reporter ion MS3 (6-plexed TMT) was turned on. The false discovery rate was
set to 1% at the level of PSMs and proteins. A score cut-off of 40 was used for identified modified peptides.
QUANTIFICATION AND STATISTICAL ANALYSIS
The abundances of TMT were log2-transformed and further analyzed by Perseus (Tyanova et al., 2016b) for statistical evaluation such
as principal component analyses and t tests. The PSP logo generator (Hornbeck et al., 2015) was used for sequence motif analysis.
DAVID (Huang da et al., 2009) was used for gene ontology and pathway enrichment analysis. STRING v11 (Szklarczyk et al., 2019)
was used for protein-protein interaction analysis. SigmaPlot (Systat Software), was used for preparing box plots.
Cell Reports Methods 2, 100138, January 24, 2022 e3
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